Pseudomonas putida is a bacterium which can grow on d-camphor as its source of carbon and energy. In the presence of camphor this organism contains a soluble cytochrome P-450-type of monooxygenase system. The membrane-bound electron transport system which occurs in this organism is unusual in that the terminal oxygen reducing enzyme is not a heme a type of cytochrome. The ultimate goal of this research program is to understand the mechanism of oxygen reduction by these two systems and their interaction. Research goal for the coming year: A) Studies of the sequence of electron carriers in the membrane-bound electron transport system of P. putida will be carried out using the stopped-flow spectrophotometer which was developed for the study of turbid suspensions. The interactions of the cytochrome P-450 system and the membrane bound electron transport system will also be studied using whole cells and sensitive spectrophotometric and fluorimetric techniques to the monitor the states of these enzymes during the steady state of oxygen consumption and camphor metabolism. B) The cytochrome P-450 dependent oxidation of camphor requires an electron transport chain consisting of a flavoprotein and an iron-sulfur protein. The proposed research will investigate the interaction of these proteins and cytochrome P-450 during electron transfer using a freeze-quench technique to prepare samples for EPR spectroscopy. Using this technique we hope to investigate the process of electron transfer to cytochrome P-450 and the "effector" roles of the electron transfer proteins in modifying and controlling this reaction. C) The studies of substrate binding to cytochrome P-450 have shown how the buffer medium can have a marked influence on the reaction at room temperature. Studies of substrate interaction with cytochrome P-450 using EPR spectroscopy are done at less than 170 degrees C. During the coming year, we will attempt to establish a firm basis for the correlation of results obtained at room temperature and at low temperature.